Suspension composition for hematology analysis control

ABSTRACT

A suspension composition for a hematology analysis control particularly useful for preserving relevant detectable characteristics of blood cells for a prolong stability period. The suspension may include at least one polysaccharide, which may include or derive from chitosan and/or chitin, as a stabilizing agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase of International ApplicationNo. PCT/US17/44368, filed Jul. 28, 2017, which claims the prioritybenefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.62/454,224, filed Feb. 3, 2017 and U.S. Provisional Application No.62/368,676, filed Jul. 29, 2016, the disclosures of all of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The teachings relates generally to hematology analysis and moreparticularly to suspension compositions for use in a synthetic control(and resulting control compositions), and a synthetic control, for asemi-automated or automated digital imaging hematology analyzers.

BACKGROUND

For decades, the traditional semi-automated or automated approach tohematology analysis has involved flow based techniques. Blood sampleshave been aspirated into a hematology analyzer and passed through adetection cell. Relevant characteristics of the blood cells have beenmeasured by light scatter characteristics, electrical characteristics,or otherwise. Based upon detected attributes of the cell, the cell willbe classified according to its size and the presence of granular nuclearmaterial. In this manner blood cell differentials can be determined forsub-populations of white blood cells.

In accordance with clinical laboratory standards, performance qualityand consistent operation of hematology instruments has employed the useof controls typically have been made from blood cell sources that aretreated to preserve the relevant detectable characteristics of thecells. Control manufacturers would use either human or animal bloodcells as a blood cell source and process them to achieve and retain asize, density and/or morphology representative of a typicalsubpopulation of cells, as detected by an instrument detectiontechnique. The respective simulated subpopulations could then becombined in a single control composition, optionally with non-leukocyteblood cell components, such as platelets, reticulated platelets, redblood cells, nucleated red blood cells, reticulocytes, immaturereticulocytes, or otherwise.

Examples of teachings addressing approaches to making simulated bloodcomponents are described, without limitation, in U.S. Pat. Nos.4,160,644 and 4,436,821 (teaching simulated platelets); U.S. Pat. No.6,221,668 (teaching simulated reticulocytes, reticulated platelets andnucleated red blood cells); U.S. Pat. No. 5,432,089 (teaching simulatedreticulocytes, and that such simulated reticulocytes may be derived froman anemic animal source); U.S. Pat. No. 6,723,563 (teaching simulatednucleated red blood cells); US Published Application No. 20120308985(teaching simulated immature reticulocytes); The resulting processedcells after transformation from their natural state, would be suspendedin a suspension composition adapted to help preserve the processed cellsand enable proper function within an instrument. It was a commonpractice to suspend the cells in a medium that included lipoprotein.This is described, for example, in U.S. Pat. Nos. 5,270,208 and5,262,327.

Examples of patents describing the processing of cells from a non-humancell source to form white blood cell subpopulation analogs include U.S.Pat. No. 5,512,485; see also, U.S. Pat. No. 4,704,364. Other examples ofteachings of making simulated blood components include United StatesPatent Application 20030104631; U.S. Pat. Nos. 6,653,063; 6,514,763; and5,858,790.

In recent years, efforts have been devoted toward development ofsemi-automated or automated hematology analyzers that employ digitalimaging to analyze patient blood. Assuring instrument integrity andconsistency of results remains a challenge for instrument producers andoperators. As with traditional semi-automated or automated hematologyinstruments, the digital imaging instruments require regular qualitycontrol testing using one or more convenient and accessible controlcompositions that yield consistent and reproducible results for aprolonged period of time, and well beyond the useful life of fresh wholeblood (e.g., fresh human whole blood).

Due to different detection techniques employed as between traditionalsemi-automated or automated hematology analyzers and digital imaginghematology analyzers, conventional hematology controls are notnecessarily useful with digital imaging hematology analyzers. There isthus a need for new compositions for use in a control for semi-automatedor automated digital imaging hematology analyzers. There is a need for anew composition that that provides relatively long term stability ofsimulated white blood cells (e.g., at least about 3 days, 7 days, 14days, 30 days, 45 days, 90 days or longer, such as at least about 105days) when stored at about 2 to about 10° C. There is a need also for anew composition that allows simulated blood cells to be dispensed (e.g.,printed) onto a substrate for delivering the simulated blood cells forsemi-automated or automated analysis by digital imaging while on thesubstrate.

SUMMARY

In general, the present teachings address one or more of the above needsby providing a synthetic control composition for a semi-automated orautomated digital imaging hematology analyzers. The present teachingsaddress one or more of the above needs by providing a unique suspensioncomposition into which simulated blood cells (e.g., cells that includesimulated nucleated blood cells) can be dispersed to form a syntheticcontrol composition for a semi-automated or automated hematologyanalyzer, and particularly a digital imaging hematology analyzer. Inthis regard, the present teachings also relate generally to syntheticcompositions for assuring quality control of a digital imaginghematology analyzer. The compositions generally will include one or moresimulated blood components (which may be derived from one or moreprocessed blood cells) and a suspension composition in accordance withthe present teachings. The suspension component is such that stabilityof the one or more simulated blood components is prolonged significantlyas compared with fresh whole blood (e.g., fresh human whole blood).

The teachings herein relate as well to a synthetic control compositionthat includes, in a unique suspension composition, one or anycombination of components for simulating a platelet, a reticulatedplatelet, a red blood cell, reticulocyte, an immature reticulocyte, anucleated red blood cell, or a simulated leukocyte population orsub-population and is useful as a quality control composition for adigital imaging hematology analyzer. The synthetic control of theteachings may be useful as a quality control composition for a digitalimaging hematology analyzer for analyzing a three-part leukocytedifferential, a five-part leukocyte differential, and/or an expandeddifferential leukocyte analysis (also referred to as an expandeddifferential white blood cell (“dWBC”) analysis).

It can be seen that the synthetic control composition of the teachingsmay include simulated leukocytes (which may be derived from a human orother source, as discussed herein) provided as an individualsubpopulation of leukocytes, and/or a collection of cells capable ofdifferentiation into at least the three and/or the five traditionalsubpopulations of leukocytes, and/or the simulated leukocytes may besuitable for providing an expanded differential white blood cell(“dWBC”) analysis and for total white blood cell count or cell count forany white blood cell sub population.

With attention now to certain other generalities about the presentteachings, it can be said that the teachings herein relate generally toa composition adapted for use in assuring quality control of a digitalimaging hematology analyzer, including a suspension medium with which atleast one simulated nucleated blood component is mixed in apredetermined amount, the suspension medium including a stabilizingagent capable of preserving relevant detectable size and morphology,including detectable nuclear morphological characteristics of thenucleated blood component (including any native nuclear cytoplasmgranules), when stored at about 2 to about 10° C. for a period of atleast 3 days, 7 days, 14 days, 30 days, 45 days, 90 days or longer, suchas at least about 105 days) from the time when the at least onesimulated nucleated blood component is initially mixed with thesuspension medium.

The control composition in which the suspension composition of theteachings is useful may be a synthetic control composition forsimulating one or more components of blood (e.g., human whole blood).The control composition in which the suspension composition of theteachings is useful may be a control composition for simulating cells ofa leukocyte population of whole blood (e.g., simulated nucleated bloodcells to resemble a leukocyte population or one or more subpopulations).Examples of a leukocyte population of whole blood for use in a controlcomposition with the present suspension composition include a three-partleukocyte population of whole blood (e.g., the control may be tosimulate three leukocyte subpopulations of whole blood (namely,neutrophils, lymphocytes and monocytes)), a five-part leukocytepopulation of whole blood (e.g., the control may be to simulate thetraditional five leukocyte subpopulations of whole blood (namely,neutrophils, eosinophils, basophils, lymphocytes, and monocytes)),and/or an extended leukocyte population of whole blood (e.g., thecontrol may include further subpopulations of one or more of thetraditional five leukocyte subpopulations of whole blood). An example ofanother simulated nucleated cell component is a nucleated red bloodcell. The suspension composition of the present teachings are alsouseful to preserve simulated reticulocytes, an immature reticulocytefraction, or both.

The suspension composition may be employed in a synthetic controlcomposition. The suspension composition may be employed in a syntheticcontrol composition in combination with simulated blood cells (e.g.,simulated nucleated blood cells, such as a simulated leukocytepopulation or sub-population of whole blood and/or a simulated nucleatedred blood cell). A simulated leukocyte population or subpopulation ofwhole blood, for the present teachings, may be derived at leastpartially, or entirely from leukocytes of human whole blood. Thesimulated leukocyte population or subpopulation of whole blood mayinclude cells that have been treated in a manner to stabilize theirrespective cell membranes so that the cells remain substantially intactfor a period of time that is longer than cells that are not stabilized.It is possible that all simulated leukocytes of a leukocyte population,including the respective subpopulations, are treated simultaneously(e.g., according to a sequence of one or more partial or complete fixingand lysing steps) to provide the simulated leukocytes.

The suspension composition may be employed in a control composition, incombination with one or more components for simulating a platelet, areticulated platelet, a red blood cell, reticulocyte, an immaturereticulocyte, a nucleated red blood cell, or any combination thereof.For example, the suspension composition may be employed in a controlcomposition in combination with a simulated nucleated red blood cellprepared from a non-human source (e.g., nucleated blood cells of a birdsuch as a turkey, a fish, or a reptile such as an alligator). Forexample, it may be possible to provide a source of blood cells suitablefor simulating a nucleated red blood cell. Provided blood cells mayindividually include a membrane enclosing a nucleus and cytoplasm. Themembrane may be stabilized to retain the nucleus and cytoplasm. Forexample, there may be a step of lysing the cells that are provided(e.g., with a solution including saponin) and then fixing with analdehyde (e.g., glutaraldehyde and/or formaldehyde). It is also possiblethat no lysing step would be utilized prior to cell fixation.

The amounts of simulated cells in the suspension composition generallyare predetermined; for example, the amounts may be known amounts forsimulating normal amounts in whole blood and/or abnormal amounts). Thesuspension composition may be useful in a stand-alone controlcomposition, in which typically a single simulated blood cell componentis employed.

In contrast with prior control compositions, the control composition ofthe present teachings need not necessarily employ a lipoprotein to helpassure proper characterization and differentiation of simulatedleukocyte cells into their proper subpopulations. The present teachingsmake use of the recognition that certain stabilization agents can bemixed in a suspension composition with a volume of simulated white bloodcells, and for a prolonged period (e.g., at least about 3 days, 7 days,14 days, 30 days, 45 days, 90 days or longer, such as at least about 105days, when stored at about 2 to about 10° C.) from the time of mixing,simulated cell components (including the simulated leukocytes) retainrelevant detectable size and morphology, including detectable nuclearmorphological characteristics of the cell and its nuclear matter,including native nuclear cytoplasm granules. However, it is alsopossible that lipoprotein may be utilized.

According to one general aspect of the teachings applicable to allembodiments, there is contemplated a suspension composition forsimulated leukocytes (e.g., cells that have similar detectablecharacteristics, such as size, a nucleus morphology and/or morphology ofother native nuclear cytoplasm granules, of one or any combination ofsub-populations of whole blood) of a hematology analyzer controlformulation. The suspension composition is especially useful for acontrol for a semi-automated or automated digital imaging analyzer. Thesuspension composition is adapted to retain the detectablecharacteristics of the simulated blood cells (e.g., simulated nucleatedcells such as nucleated leukocytes and/or nucleated red blood cells)over a prolonged period of storage. The suspension composition mayinclude a buffered aqueous solution.

As to all embodiments, the suspension composition may include at leastone stabilizing agent (e.g., one that includes at least onepolysaccharide, such as (without limitation) a nitrogen-containingpolysaccharide) having a polymerization degree ranging from greater thanone to about 100). The at least one stabilizing agent may be present inan amount sufficient for preserving stability of the detectable size andmorphological characteristics of the simulated simulated blood cells(e.g., simulated nucleated cells such as nucleated leukocytes and/ornucleated red blood cells) for a period of at least about 3 days (e.g.,at least about 3 days, 7 days, 14 days, 30 days, 45 days, 90 days orlonger, such as at least about 105 days) when stored at about 2 to about10° C.), from the time of suspending the simulated cells.

Generally, the suspension composition may be adapted (as to allembodiments) for use in digital imaging hematology instrument thatcreates and analyzes an image, such as by applying one or more automatedor semi-automated analytical techniques, after a sample has beendispensed onto a substrate. For instance, upon mixing the suspensioncomposition with the simulated leukocytes, the resulting mixture iscapable of dispensing through a nozzle for delivery to a substrate foranalysis by a digital imaging hematology analyzer. The suspensioncomposition may be adapted, upon mixing with the simulated blood cells(e.g., simulated nucleated cells such as nucleated leukocytes and/ornucleated red blood cells), for dispensing through a nozzle (e.g., acapillary or other tube) for delivery by printing to a transparentsubstrate (e.g., a glass or polymeric slide) and subsequent analysis bya semi-automated or automated digital imaging hematology analyzer,without any material damage (e.g., damage to excess of five percent(10%) by number of total simulated cells) to the simulated cells. It ispossible that the control composition may be delivered into a cassetteor cartridge device which may provide digital images for identifyingcell populations.

In general, as to all embodiments, the aqueous buffered solution of thesuspension composition may include at least one buffering agent.

The aqueous buffered solution may include at least one dispersion agentfor reducing aggregation of the simulated blood cells (e.g., simulatednucleated cells such as nucleated leukocytes and/or nucleated red bloodcells) as compared with the aqueous buffered solution without thedispersion agent.

The suspension composition may have a pH ranging from about 6 to about8.

The at least one stabilizing agent may include an organic compoundhaving at least one glycosidic linkage. The at least one stabilizingagent may include a compound that includes an amine moiety and acarbohydrate (e.g., glucose and/or dextrose) moiety.

The at least one stabilizing agent may include at least one aminopolysaccharide having a polymerization degree ranging from greater thanone to about 100 (e.g., from greater than about 5 to about 40). The atleast one stabilizing agent may include or consist of an oligosaccharide(or a derivative of an oligosaccharide) having a weight averagemolecular weight (measured by high performance liquid chromatography,which may be further verified by comparison with commercially availablestandards)) from about 100 to about 15,000 daltons (Da), from about 250to about 10,000 Da, about or even about 1000 to about 3000 Da.

The at least one stabilizing agent may include a glucosamine, or aderivative thereof. It may include one or more of a chitosan and/orchitin, a salt of a chitosan and/or chitin, and/or some other derivativeof a chitosan and/or chitin.

The at least one stabilizing agent may be in a polymeric form. The atleast one stabilizing agent may be in a salt form (e.g., a salt of achitosan and/or chitin). Illustrative salts include one or anycombination of a citrate, a malate, a lactate, an acetate, a formate, aglyoxylate, a pyruvate, an ascorbate or glycolate.

Generally applicable to all embodiments, the at least one stabilizingagent may be present in an amount up to about ten percent (15%) (e.g.,up to about ten percent (10%) or about seven percent (7%)) of thesuspension composition. The at least one stabilizing agent may bepresent in an amount up to about ten percent (10%) (e.g., up to aboutseven percent (7%) or about five percent (5%) of a resulting controlcomposition admixture including the suspension composition and thesimulated cells.

Unlike certain control compositions of the prior art, the suspensioncomposition and any resulting control composition employing thesuspension composition of the general teachings herein may besubstantially free of any added lipid (e.g., lipoprotein), anyglycoprotein or both.

The teachings herein also contemplate a blood control composition (e.g.,leukocyte and/or nucleated red blood cell-containing control)composition adapted for use in a semi-automated or automated digitalimaging hematology analyzer comprising the suspension composition of theteachings, as well as the use of the composition. For instance, theteachings envision a method of using the suspension compositionincluding a step of dispensing the suspension composition onto asubstrate, and at least partially evaporating water from the aqueousbuffered suspension composition. Thereafter, a digital image can bemade. For instance, when cells are deposited onto a substrate with thesuspension composition, a digital image may be made of the cells. Theimage may be analyzed. For instance the image may be analyzed by acomputer implemented technique.

Other benefits and advantages of the teachings will be understood uponreview of the remaining teachings, which provide additional details.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a micrograph illustrating cells of fresh human whole blood, asviewed by optical microscopy (oil immersion lens at 100×), in a form aswould be detectable by a digital imaging hematology analyzer.

FIG. 2 is a micrograph illustrating simulated leukocytes cells in afreshly prepared suspension composition of the present teachings asviewed by optical microscopy (oil immersion lens at 100×), in a form aswould be analyzed by a digital imaging hematology analyzer.

FIG. 3a is an enlarged micrograph illustrating respective examples ofsimulate leukocyte subpopulation cells and other simulated cellcomponents in a suspension composition of the present teachings asviewed by optical microscopy (oil immersion lens at 100×), in a form aswould be analyzed by a digital imaging hematology analyzer.

FIG. 3b is an enlarged micrograph illustrating respective examples ofleukocyte subpopulation cells and other simulated cell components afterstorage at about 2 to about 10° C. for 105 days in a suspensioncomposition of the present teachings as viewed by optical microscopy(oil immersion lens at 100×), in a form as would be analyzed by adigital imaging hematology analyzer.

FIG. 4a is a micrograph to illustrate an example of simulated leukocytecells in a suspension medium including a stabilizing agent of thepresent teachings after about three weeks as viewed by opticalmicroscopy (oil immersion lens at 100×), in a form as would be analyzedby a digital imaging hematology analyzer.

FIG. 4b is a micrograph to illustrate an example of simulated leukocytecells of the present teachings in a suspension medium as in FIG. 4a ,but absent any stabilizing agent of FIG. 4a after about three weeks asviewed by optical microscopy (oil immersion lens at 100×), in a form aswould be analyzed by a digital imaging hematology analyzer.

FIG. 5 is a micrograph to illustrate an example of a prior arthematology control, absent any stabilizing agent of the presentteachings, as viewed by optical microscopy (oil immersion lens at 100×),in a form as would be analyzed by a digital imaging hematology analyzer.

DETAILED DESCRIPTION

The explanations and illustrations presented herein are intended toacquaint others skilled in the art with the teachings, its principles,and its practical application. Those skilled in the art may adapt andapply the teachings in its numerous forms, as may be best suited to therequirements of a particular use. Accordingly, the specific embodimentsof the present teachings as set forth are not intended as beingexhaustive or limiting of the teachings. The scope of the teachingsshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to theappended claims, along with the full scope of equivalents to which suchclaims are entitled. The disclosures of all articles and references,including patent applications and publications, are incorporated byreference for all purposes. Other combinations are also possible as willbe gleaned from the following claims, which are also hereby incorporatedby reference into this written description.

This application claims the benefit of the filing date of U.S.Provisional Application Ser. No. 62/368,676, filed Jul. 29, 2016 and62/454,224, filed Feb. 3, 2017, the contents of these applications beinghereby incorporated by reference herein for all purposes.

Unless otherwise stated, concentrations are expressed in weight/volumepercentages (w/v %). Thus, to illustrate, 1% of an ingredient in aliquid medium would refer to 1 gram of the ingredient in 100 millilitersof the liquid. Unless otherwise stated, references herein to “wholeblood” and/or “blood cells”, include human whole blood. Though theteachings herein are particularly applicable in human whole bloodanalysis, they are not so limited, and may have application inveterinary applications as well. The phrase “digital imaging hematologyinstrument” refers to automated hematology analyzers of an automated orsemi-automated type that employ computer implemented analysis of adigital image of a sample (e.g., a computer may analyze the sample toascertain cell size, cell morphology, count cells, and/or classifycells, and may also output resulting data and/or images for independenthuman analysis). An example of such analysis technology is exemplifiedby the Cobas m511™, by Roche Diagnostics, which employs what is referredto as Bloodhound™ analysis technology. It is believed that suchtechnology is described in one or more of U.S. Patent Application Nos.20090269799; 20110014645; 20130070077; 20130077085; and WO/2013/016038.

The phrase “detectable characteristics” refers to one or morecharacteristics (e.g., a physical characteristic, such as size and/ormorphology) that are detectable by a hematology instrument, whether adigital imaging hematology instrument or otherwise.

In general, the teachings herein contemplates a suspension compositionfor preserving the stability of simulated blood cells of a hematologycontrol composition. The teachings more particularly contemplates asuspension composition for preserving the stability of simulated bloodcells, so that the cells exhibit and retain relevant detectablecharacteristics, such as size, and/or morphology. One particularadvantage of the teachings is the ability of the suspension compositionto preserve the stability nuclear morphological characteristics(including morphology of native nuclear cytoplasm granules) for a periodsubstantially longer than fresh human whole blood. The suspensioncomposition teachings are useful to prepare a suspension of a controlcomposition for assuring quality of a hematology analyzer (e.g., adigital imaging hematology analyzer).

In general, the suspension composition is useful for and may be part ofa control composition that include the suspension and simulated bloodcells (e.g., simulated nucleated cells such as nucleated leukocytesand/or nucleated red blood cells). Such control composition and methodsof using the controls are part of the teachings herein as well.

Determination of whether relevant detectable characteristics areretained can be done by optical examination (e.g., including the use ofa digital imaging device for outputting an image) of a statisticallysignificant number of samples, with analysis to identify materialdeviations from native morphological attributes of the type of cellunder consideration. Indicators of material deviations can employoptical means to identify. It may include a comparative study of asample with data (which may include images) about known characteristicsof the sample material in its normal freshly prepared state. The skilledperson likewise would recognize that relevant detectable characteristicshave not been attained, or are no longer retained (e.g., a stabilityperiod has been exceeded) when a sample intended yields cell counts thatdeviate from the intended values in excess of at least ten percent(10%), or there is some other evidence indicative of an inability todifferentiate cells; when nucleated blood cells present identifiablesurface cracks in images at a magnification of at least 100× (e.g., whenviewed by optical microscopy with an oil immersion lens); when cellularcytoplasm appears de-granulated and not readily identifiable in imagesat a magnification of at least 100× (e.g., when viewed by opticalmicroscopy with an oil immersion lens), and/or when one or more nucleisizes deviate from their normal well known sizes.

It should thus be recognized, that the phrase “simulated blood cells” isused herein not only to refers to cells from a non-human source that areadapted to resemble blood cells from a human (or those of anotherspecies) in relevant detectable characteristics by a hematology analyzer(e.g., a digital imaging analyzer). The phrase “simulated blood cells”also refers to blood cells that are true human blood cells. Thus, asource of simulated white blood cells, in accordance with the teachingsherein may be human white blood cells. Moreover, any of the simulatedblood cells that are suspended in the suspension composition may becells derived from human whole blood and/or non-human whole blood source(e.g., blood from a source selected from avian blood, fish blood,reptilian blood, mammalian blood or otherwise.

The suspension composition may be employed in a control composition,alone or in combination with simulated blood cells, which may includesimulated leukocytes or other simulated cell components of whole blood(e.g., simulated nucleated cells such as nucleated leukocytes and/ornucleated red blood cells). A simulated leukocyte population orsubpopulation of whole blood may be derived at least partially, orentirely from leukocytes of human whole blood. The simulated leukocytepopulation or subpopulation of whole blood may include cells that havebeen treated in a manner to stabilize their respective cell membranes sothat the cells remain substantially intact for a period of time that islonger than cells that are unstabilized. It is possible that allsimulated leukocytes of a leukocyte population, including the respectivesubpopulations, are treated simultaneously to provide the simulatedleukocytes. For example, a source of leukocytes (e.g., a leukocyte packfrom a blood bank) may be provided and the leukocytes contacted with astabilizer to partially stabilize the cell membranes of the leukocytes.The source is then lysed to eliminate red blood cells. The leukocytesmay then be further stabilized prior to combining them with thesuspension.

In general, the present teachings provide a unique suspensioncomposition into which simulated blood cells can be dispersed to form acontrol composition. In contrast with prior control compositions, thecontrol composition of the present teachings need not necessarily employa lipoprotein to help assure proper characterization of simulatedleukocytes and/or differentiation of the simulated blood cells intotheir proper subpopulations.

The present teachings make use of the recognition that certainstabilization agents can be mixed in a suspension composition with avolume of simulated blood cells (e.g., for simulation one or moresubpopulations of white blood cells), and for a prolonged stabilityperiod (e.g., at least about 3, at least about 7, at least about 14, atleast about 30, at least about 45, at least about 90 days or longer,e.g., at least about 105 days, while maintained at a temperature ofabout 2 to about 10° C.) from time of mixing, the simulated blood cells(e.g., simulated leukocytes, and any other simulated blood cellcomponent such as a nucleated red blood cell component) retain relevantdetectable characteristics such as size and/or nuclear morphologicalcharacteristics (including morphology of native nuclear cytoplasmgranules).

According to one general aspect of the teachings applicable to allembodiments, there is contemplated a suspension composition forsimulated blood cells of a hematology analyzer control formulation,which may be a digital imaging hematology analyzer. The simulated bloodcells may be cells from a suitable source that have been processed toattain and/or preserve characteristics detectable by a hematologyanalyzer, such as a digital imaging hematology analyzer. For instance,the characteristics that are attained and/or preserved may be size, anucleus morphology and/or morphology of other nuclear cytoplasmgranules.

The teachings herein have application for suspending and preservingstability of simulated blood components (e.g., blood cells) in a controlcomposition. For example, the teachings have application for suspendingand preserving stability of simulated leukocytes of a controlcomposition. The simulated leukocytes (which may be derived from a humanor other source, as discussed above) may be provided as an individualsubpopulation of leukocytes, and/or a collection of cells capable ofdifferentiation into at least the three and/or the five traditionalsubpopulations of leukocytes. The simulated leukocytes may be suitablefor providing an expanded differential white blood cell (“dWBC”)analysis.

A control in accordance with the teachings for an expanded dWBC analysismay include (e.g., in addition to the traditional 3 or 5 subpopulations)components that simulate one or more cell populations that may includeblasts, immature granulocytes, atypical lymphocytes (types I, II andIII), myeloid precursors (myeloblast, promyelocyte, myelocyte andmetamyelocyte) and or lymphocyte subsets (T-Cells and B-Cells).

The suspension composition is adapted to retain the detectablecharacteristics of the simulated blood cells (e.g., white blood cells)over a prolonged period of storage. For example, the suspensioncomposition (whether used for simulated white blood cells and/or otherblood cells is formulated to preserve relevant detectablecharacteristics, such as size and/or nuclear morphologicalcharacteristics (including morphology of native nuclear cytoplasmgranules) of the simulated blood cells, for a prolonged stability period(e.g., at least about 3, at least about 7, at least about 14, at leastabout 30, at least about 45, at least about 90 days or longer, e.g., atleast about 105 days) when stored at about 2 to about 10° C. from timeof mixing with the suspension composition.

Throughout the prolonged stability period, the nucleated cells (e.g.,the simulated leukocytes (and/or any other simulated cell componentssuch as simulated nucleated red blood cells) will resemble to a detectorof a hematology analyzer, and particularly a digital imaging hematologyanalyzer (e.g., without limitation, a COBAS m511™ analyzer from Roche)the blood cell it is intended to simulate. Thus, the simulated bloodcell is capable of being subjected to sample processing by a digitalimaging hematology instrument, during which the cell may be deposited ona slide (such as by a printing operation), stained, and imaged in amanner suitable for computer implemented image analysis.

The suspension composition may be isotonic. The suspension compositionmay include a buffered aqueous solution. The suspension composition mayinclude a mixture of cell stabilizers. The suspension composition mayinclude an antimicrobial. The suspension composition may include one ormore agents for maintaining a predetermined pH.

As to all embodiments, the suspension composition may include at leastone stabilizing agent (e.g., one that includes at least onepolysaccharide having a polymerization degree ranging from greater thanone to about 100, such as (without limitation) a nitrogen containingpolysaccharide). The at least one stabilizing agent may be present in anamount sufficient for preserving stability of the detectablemorphological characteristics of the white blood cells for a period ofat least about 3 days (e.g., at least about 7, at least about 14, atleast about 30, at least about 45, at least about 90 days or longer,e.g., at least about 105 days), after being stored during such period atabout 2 to about 10° C., upon suspending the simulated white bloodcells.

Generally, the suspension composition may be adapted (as to allembodiments) for use in digital imaging hematology instrument thatcreates and analyzes by a computer implemented technique an image of asample that has been dispensed onto a substrate. For instance, uponmixing the suspension composition with the simulated leukocytes, theresulting mixture is capable of dispensing through a nozzle for deliveryto a substrate for analysis by a digital imaging hematology analyzer.The suspension composition may be adapted, upon mixing with thesimulated white blood cells, for dispensing through a nozzle (e.g., acapillary or other tube) for delivery by printing to a transparentsubstrate (e.g., a glass or polymeric slide) and subsequent analysis bya digital imaging hematology analyzer, without any material damage(e.g., damage to excess of five percent (10%) by number of totalsimulated blood cells) to the simulated blood cells.

The at least one stabilizing agent may be present (as to allembodiments) in an amount sufficient for preserving stability of thedetectable morphological characteristics of the white blood cells for aperiod of at least 30 days, after being stored during such period atabout 2 to about 10° C., upon suspending the white blood cells. Morespecifically, as applicable to the teachings in general, the at leastone stabilizing agent may function to retain intact relevant detectablecharacteristics such as size and/or nuclear morphologicalcharacteristics (including morphology of native nuclear cytoplasmgranules) of one or more of any simulated nucleated blood cell componentof a control composition. Thus, the size and shape of a nucleus of thesimulated blood cell be preserved substantially as it would be in itsnative state in fresh whole blood. It is also possible that the size,shape and/or amount of other nuclear matter (e.g., cytoplasm) may bepreserved substantially as it would be in its native state in freshwhole blood.

The at least one stabilizing agent may be part of a stabilizationmixture. Such stabilization mixture may include two, three, four, fiveor more ingredients. Such stabilization mixture may include twelve, ten,eight or fewer ingredients. The ingredients of the stabilization mixturemay be in an amount effective for stabilizing one or more aspects of asimulated blood cell (e.g., a simulated leukocyte or any other simulatedblood cell component, such as a platelet, a reticulated platelet, a redblood cell, reticulocyte, an immature reticulocyte, a nucleated redblood cell, or any combination thereof). For example, one or more of theingredients may be for stabilizing a membrane, for stabilizing nuclearmaterial, for stabilizing a nucleic acid, for stabilizing cytoplasm, orotherwise).

In general, as to all embodiments, the aqueous buffered solution of thesuspension composition may include at least one buffering agent. It mayinclude at least one antimicrobial. The aqueous buffered solution mayinclude at least one dispersion agent for reducing aggregation of thesimulated white blood cells as compared with the aqueous bufferedsolution without the dispersion agent. The suspension composition mayhave a pH ranging from about 6 to about 8.

In general, as to all embodiments, with regard to the at least onestabilizing agent, it may include at least one polysaccharide, such asis set forth in the following. The at least one stabilizing agent may bea nitrogen-containing polysaccharide. The at least one stabilizing agentmay be a derivative of glucose. It may include one or more glucosemoieties. However, the at least one stabilizing agent may besubstantially free of simple glucose (i.e., that represented by theformula C₆H₁₂O₆); that is, the suspension medium may include less thanabout 10%, or less than about 5% glucose of the suspension medium.

For example, the at least one stabilizing agent may include at least oneamino polysaccharide having a polymerization degree ranging from greaterthan one to about 100 (e.g., from greater than about 5 to about 40). Theat least one stabilizing agent may include or consist of anoligosaccharide (or a derivative of an oligosaccharide) having a weightaverage molecular weight (measured by high performance liquidchromatography, which may be further verified by comparison withcommercially available standards)) from about 250 to about 10,000daltons (Da), from about 250 to about 10,000 Da, or even about 1000 toabout 3000 Da.

The at least one stabilizing agent may include a glucosamine, or aderivative thereof. It may include one or more of a chitosan, a salt ofa chitosan or some other derivative of a chitosan.

The at least one stabilizing agent may be in a polymeric form. The atleast one stabilizing agent may be in a salt form (e.g., a salt of achitosan). Illustrative salts include one or any combination of acitrate, a malate, a lactate, an acetate, a formate, a glyoxylate, apyruvate, an ascorbate or glycolate.

By way of example, the at least one stabilizing agent may include one orany combination of D-(+)-glucosamine, N-acetyl-D-glucosamine, chitosanacetate, chitosan lactate, chitosan oligosaccharide, chitin, carboxylmethyl chitosan, a derivative of any of these listed agents, or anycombination of the same.

Generally applicable to all embodiments, the at least one stabilizingagent may be present in an amount up to about fifteen percent (15%)(e.g., up to about ten percent (10%) or about seven percent (7%)) aboutten percent (10%) or about seven percent (7%)) of the suspensioncomposition. The at least one stabilizing agent may be present in anamount up to about ten percent (10%) (e.g., up to about seven percent(7%) or about five percent (5%) of a resulting control compositionadmixture including the suspension composition and the white bloodcells. Generally applicable to all embodiments, the at least onestabilizing agent along with any other ingredients functional forpreserving stability of a simulated blood cell (i.e., the totalfunctional ingredients of the stabilizing mixture), may be present inamount up to about twenty five percent (25%); twenty two percent (22%);or about eighteen (18%) (e.g., up to about fifteen percent (15%) orabout ten percent (10%)) of the suspension composition. The totalfunctional ingredients of the stabilizing mixture may be present in anamount of at least about one percent (1%) (e.g., at least about sevenpercent (3%) or about five percent (5%)) of a resulting controlcomposition admixture including the suspension composition and the whiteblood cells.

Unlike certain control compositions of the prior art, the suspensioncomposition and any resulting control composition employing thesuspension composition of the general teachings herein may besubstantially free of any added lipid (e.g., lipoprotein), anyglycoprotein or both. For example, it may have less than 0.3 percent(e.g., less than about 0.1 percent) of a lipid and/or glycoprotein.Alternatively stated, to the extent any lipid (e.g., lipoprotein), anyglycoprotein or both are employed in a suspension composition inaccordance with the present teachings, such lipid (e.g., lipoprotein),glycoprotein or both is employed in an insufficient amount to materiallyalter the stability characteristics of any of the resulting bloodcomponents of the resulting control composition during the stabilityperiod. For instance, the amount is an amount at which detectablenuclear morphological characteristics of the nucleated blood component(including any native nuclear cytoplasm granules) exhibit less than a10%, or even less than a 5%, variation in detected cell counts whenanalyzed by a digital hematology analyzer as compared with a controlhaving no such lipid (e.g., lipoprotein), glycoprotein or both.

Though the suspension composition may be substantially free of anylipoprotein, it may optionally include lipoprotein. For instance, it mayinclude a lipoprotein in a suitable amount for assuring properdifferentiation of white blood cells added to the suspensioncomposition. The suspension composition may include, or it may be freeof an antioxidant (e.g., in an amount sufficient to prevent cell lysisduring the stability period of a control in which the suspensioncomposition is used).

The suspension composition may include at least one cellular aggregationinhibition ingredient, or may be free of any cellular aggregationinhibition ingredient. The suspension composition may include asurfactant (e.g., in an amount sufficient to effect a surface energycharacteristic of any of the simulated blood cells), or it may be freeof any surfactant. Examples of surfactants can be found in U.S. Pat. No.5,250,438, (see, e.g., column 3), incorporated by reference.

In general, as to all embodiments, the aqueous buffered solution of thesuspension composition may include at least one buffering agent. Forexample, it may be zwitterionic buffering agent. The at least onebuffering agent may include a sulfonic acid moiety. The buffering agentmay be selected from one or any combination of3-Morpholinopropane-1-sulfonic acid (MOPS);2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid(TES); or 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid(HEPES). The at least one buffering agent may be present in an amount ofup to about 7 percent, up to about 5 percent or up to about 3 percent ofthe suspension composition. The at least one buffering agent may bepresent in an amount of greater than about 0.5 percent or greater thanabout 1 percent of the suspension composition.

The suspension composition may include at least one antimicrobial. Theat least one antimicrobial may be part of the aqueous buffered solutionor a separate ingredient. The at least one antimicrobial may be organicor inorganic. The at least one antimicrobial may include a biocide, amicrobe growth inhibitor, and/or a microbe reproduction inhibitor. Theat least one anti-microbial may be a bacteriostatic ingredient (e.g.,one that inhibits cytochrome oxidase in gram negative bacteria). The atleast one anti-microbial may be an anti-fungal ingredient. The at leastone anti-microbial may be an anti-yeast ingredient. Examples of suitableantimicrobials include one or more antimicrobials selected fromchloramphenicol, sodium azide, neomycin sulfate, Rifampicin minocycline,ciproflaxin, doxycycline, sulfasalazine or the like. The at least oneantimicrobial may be present in an amount of up to about 5%, up to about3% or up to about 1% percent of the suspension composition.

The suspension composition may have a pH ranging from about 6 to about8. Suitable amounts of neutralizing agents may be employed in thesuspension composition. For example, a relatively mild acid such and/ora relatively mild base such as sodium hydroxide may be titrated into thesuspension composition as needed to achieve the desired pH level.

As gleaned from the above, it is envisioned that the suspensioncomposition may include one or more inorganic compounds. The inorganiccompound may include a metallic atom or cation, such as an atom orcation of an alkali metal, an alkaline earth metal, a transition metal,or any combination thereof. For example, it is possible that theresulting suspension composition will have one or more metal atoms orcations selected from magnesium (Mg), calcium (Ca), sodium (Na),potassium (K), lithium (Li), iron (Fe), or otherwise. The amount of themetal atoms or cations may be quantified as a proportion relative to thepolysaccharide of the suspension composition. For example, the amount ofatoms or cations by weight relative to the polysaccharide may range fromabout 0.1:1 to about 2:1 (e.g., about 0.3:1 to about 1:1).

To the extent not covered in the above, the suspension composition mayinclude one or more other ingredients, including one or any combinationof an alcohol, a surfactant; a protease inhibitor; serum albuminprotein; an anticoagulant (e.g., ethylenediaminetetraacetic acid“EDTA”), a gelatin, an aldehyde (e.g., glutaraldehyde and/orformaldehyde), a polyol (e.g., polyethylene glycol), a cellulosic agent,an antioxidant, a protein, a blood serum fraction, plasma, amino acidprecursor, a cholesterol (e.g., a lipoprotein), or any combination ofthe above.

In general, water used for the aqueous compositions herein may bedistilled water, deionized water, filtered water, any combinationthereof or another source of water that is free of detectable amounts ofcontaminants that would materially impact performance of the suspensioncomposition.

The teachings herein also contemplate a blood control composition (e.g.,a white blood cell control composition) adapted for use in a digitalimaging hematology analyzer comprising the suspension composition of theteachings, as well as the use of the composition. For instance, theteachings envision a method of using the suspension compositionincluding a step of dispensing the suspension composition onto asubstrate, and at least partially evaporating water from the aqueousbuffered suspension composition. Thereafter, a digital image can bemade. For instance, when cells are deposited onto a substrate with thesuspension composition, a digital image may be made of the cells. Theimage may be analyzed.

Turning now to the accompanying FIGS. 1, 2, 3 a, 3 b, 4 a, 4 b, and 5various benefits and advantages of the teachings can be further gleaned.Except as otherwise described, the images are believed representative ofcompositions that would result using a suspension medium, generally, ofthe present teachings, and particularly the suspension medium describedbelow in Example 1. The images are believed representative ofcompositions that would result from simulated cell components thatinclude simulated leukocytes, wherein the simulated leukocytes arederived from human blood cells. Similar results are also expected whenthe simulated leukocytes are derived from non-human blood cells.

FIG. 1 is a micrograph illustrating cells of fresh human whole blood, asviewed by optical microscopy (oil immersion lens at 100×), in a form aswould be detectable by a digital imaging hematology analyzer. Examplesof the cellular components are labeled for reference purposes. Upondraw, the respective cellular components of the fresh human whole bloodsubstantially identically resemble the cellular components as would becirculating within person from whom the blood was drawn.

FIG. 2 is a micrograph illustrating simulated leukocytes cells in afreshly prepared suspension composition of the present teachings asviewed by optical microscopy (oil immersion lens at 100×), in a form aswould be detectable by a digital imaging hematology analyzer. As seenfrom FIG. 2, there is substantial identify as between the simulatedcellular components and the cellular components of FIG. 1. In accordancewith the teachings in general, the suspension medium of the presentteachings is particularly attractive for us with simulated blood cellcomponents (e.g., at least simulated leukocytes, such as stabilizedhuman leukocytes).

FIGS. 3a and 3b are included for comparison.

FIG. 3a is an enlarged micrograph illustrating respective examples ofsimulated leukocyte subpopulation cells and other simulated cellcomponents in a suspension composition of the present teachings, thecomponents and suspension composition being freshly prepared, as viewedby optical microscopy (oil immersion lens at 100×), in a form as wouldbe detectable by a digital imaging hematology analyzer.

For comparison with FIG. 3a , FIG. 3b is an enlarged micrographillustrating respective examples of leukocyte subpopulation cells andother simulated cell components after storage at about 2 to about 10° C.for 105 days in a suspension composition of the present teachings asviewed by optical microscopy (oil immersion lens at 100×), in a form aswould be detectable by a digital imaging hematology analyzer. The imageillustrates that as between the freshly prepared materials of FIG. 3aand those of FIG. 3b , there are indistinguishable differences if any.As expected in general for the teachings herein, the nucleic subjectmatter within the granulated cells, in particular, remains intactthrough a useful life orders of magnitude longer than it would if notsuspending in the suspension medium of the teachings (e.g., at least 105days).

FIGS. 4a and 4b are included for comparison.

FIG. 4a is a micrograph to illustrate an example of simulated leukocytecells in a suspension medium including a stabilizing agent (e.g., apolysaccharide, such as chitosan or another glucosamine, having apolymerization degree ranging from greater than one to about 100) of thepresent teachings after about three weeks as viewed by opticalmicroscopy (oil immersion lens at 100×), in a form as would bedetectable by a digital imaging hematology analyzer. Consistent with theillustrations of the teachings in FIGS. 2, 3 a and 3 b, the cellularcomponents resemble those of fresh whole blood (e.g., fresh human wholeblood), as seen in FIG. 1.

FIG. 4b is a micrograph to illustrate an example of simulated leukocytecells of the present teachings in a suspension medium as in FIG. 4a ,but absent any stabilizing agent of FIG. 4a after about three weeks asviewed by optical microscopy (oil immersion lens at 100×), in a form aswould be detectable by a digital imaging hematology analyzer. As can beseen, there is substantial visible degradation. For example, at leastthe simulated monocytes show considerable transformation from theirexpected state when suspended in the suspension medium of the presentteachings.

FIG. 5 is a micrograph to illustrate an example of a prior arthematology control, absent any stabilizing agent of the presentteachings, as viewed by optical microscopy (oil immersion lens at 100×),in a form as would be analyzed by a digital imaging hematology analyzer.It can be seen how nuclear matter is not identifiable.

EXAMPLES

Other benefits and advantages of the teachings will be understood uponreview of the following Examples, which provide additional illustrativedetails of the present teachings.

Example 1

The suspension composition of Table 1 is prepared and predeterminedamounts of simulated blood cells is added into it. Water makes up thebalance. Concentrations are expressed in weight/volume percentages. Tothe suspension composition, simulated blood cell components are addedaccording to three different predetermined amounts for each to form aresulting control composition. The three amounts correspond respectivelyto three different levels—a low abnormal level (“L.1”), a normal healthylevel (“L.2”) and a high abnormal level (“L.3). A stability study isperformed. The amounts for each leukocyte sub-population reported areprovided in numbers (#) and percentage of the overall leukocytepopulation (%). Measurements are taken at the time the resulting controlcomposition, day 56 of the study and day 105 of the study. The data arecollected on a Roche Cobas m511 hematology Analyzer. After preparationof the resulting control composition, and throughout the duration of thestudy, the resulting control composition is maintained at a temperatureof from about 2 to about 10° C. The results show that the suspensionmedium contributes to stability of the simulated blood cell componentsof the resulting control composition. This is consistent with thecomparative images of FIGS. 3a and 3b .

TABLE 1 Concentration (Pre-cell Ingredient Addition)(w/v %) EDTA,Disodium 1.10% Magnesium Chloride 0.03% HEPES 1.19% Calcium Acetate0.05% Polyethylene Glycol 20,000 1.00% Urea 0.50% Sodium Chloride 2.64%Chloramphenicol 0.01% Sodium Azide 0.10% Bovine Serum Albumin 1.00%Chitosan Oligosaccharide 0.50% Sodium Hydroxide 0.18%

The composition is analyzed using a Roche Cobas m511™ analyzer, with alllevels analyzed in triplicate and reported as the average of the threeanalyses. Tests are performed on two lots of compositions. Results arereported in the below Tables 2 and 3, respectively for the first lot andthe second lot.

TABLE 2 L.1 Day# 0 56 105 L.2 Day# 0 56 105 L3 Day# 0 56 105 WBC 15.9215.67 15.33 WBC 8.00 7.45 7.77 WBC 2.40 2.17 2.25 RBC 2.25 2.33 2.35 RBC4.04 3.97 4.10 RBC 5.06 5.15 5.36 HGB 5.2 5.1 5.2 HGB 10.8 10.5 10.9 HGB16.4 16.6 17.1 HCT 15.5 15.5 15.8 HCT 31.7 30.7 31.8 HCT 46.0 46.4 48.3MCV 69.0 66.6 67.2 MCV 78.3 77.2 77.6 MCV 90.9 90.0 90.1 MCH 22.9 22.122.1 MCH 26.8 26.5 26.7 MCH 32.4 32.1 31.9 MCHC 33.1 33.2 32.8 MCHC 34.234.3 34.4 MCHC 35.7 35.7 35.4 PLT 432 453 467 PLT 198 221 204 PLT 56 6561 RDW 15.6 15.9 16.2 RDW 14.0 14.5 14.1 RDW 12.9 12.5 12.5 SD RDW 38.838.2 39.3 SD RDW 39.4 40.3 39.5 SD RDW 42.2 40.7 40.5 MPV 9.8 9.9 9.7MPV 9.5 9.2 9.3 MPV 8.9 9.2 9.3 % nRBC 0.3 0.1 0.1 % nRBC 19.5 18.6 13.6% nRBC 10.9 10.6 9.9 # nRBC 0.0 0.0 0.0 # nRBC 1.6 1.4 1.1 # nRBC 0.30.2 0.2 % RETIC 7.04 7.29 6.78 % RETIC 3.93 3.53 2.60 % RETIC 0.84 0.650.21 # RETIC 0.16 0.18 0.16 # RETIC 0.16 0.14 0.11 # RETIC 0.04 0.040.01 % NEUT 59.9 59.6 55.4 % NEUT 59.2 58.3 56.4 % NEUT 59.1 59.0 58.6 #NEUT 9.54 9.36 8.50 # NEUT 4.74 4.35 4.38 # NEUT 1.42 1.28 1.32 % LYM22.6 22.1 24.1 % LYM 22.5 23.3 23.9 % LYM 22.4 20.5 21.6 # LYM 3.60 3.463.70 # LYM 1.80 1.74 1.86 # LYM 0.54 0.45 0.49 % MONO 10.1 9.1 10.1 %MONO 12.8 12.8 13.0 % MONO 14.7 16.1 16.2 # MONO 1.60 1.43 1.55 # MONO1.03 0.96 1.01 # MONO 0.35 0.35 0.36 % EOS 7.1 8.5 9.7 % EOS 5.0 4.7 6.1% EOS 3.5 3.9 3.2 # EOS 1.12 1.33 1.48 # EOS 0.40 0.35 0.47 # EOS 0.080.08 0.07 % BASO 0.4 0.7 0.7 % BASO 0.4 0.8 0.7 % BASO 0.2 0.5 0.5 #BASO 0.06 0.10 0.10 # BASO 0.03 0.06 0.05 # BASO 0.01 0.01 0.01

TABLE 3 L.1 Day# 0 52 105 L.2 Day# 0 52 105 L.3 Day# 0 52 105 WBC 18.6019.29 18.90 WBC 9.20 9.18 9.12 WBC 2.71 2.85 2.67 RBC 2.48 2.52 2.44 RBC4.29 4.35 4.18 RBC 5.36 5.54 5.42 HGB 5.9 6.1 5.9 HGB 11.7 11.8 11.4 HGB17.9 18.4 18.0 HCT 17.5 18.0 17.4 HCT 33.5 33.9 33.1 HCT 49.4 50.8 50.1MCV 70.7 71.3 71.1 MCV 78.1 78.0 79.1 MCV 92.2 91.6 92.4 MCH 23.9 24.224.2 MCH 27.2 27.1 27.3 MCH 33.4 33.2 33.4 MCHC 33.8 34.0 34.0 MCHC 34.934.8 34.6 MCHC 36.2 36.2 36.1 PLT 576 583 559 PLT 261 272 252 PLT 76 8180 RDW 14.4 13.8 13.8 RDW 12.4 11.6 11.6 RDW 10.6 9.4 9.9 StDev RDW 36.735.2 35.3 StDev RDW 34.8 32.6 33.0 StDev RDW 35.1 30.9 33.0 MPV 8.6 8.58.1 MPV 8.7 8.4 8.1 MPV 8.7 8.7 8.3 % nRBC 0.0 0.1 0.0 % nRBC 20.2 19.518.8 % nRBC 10.8 10.5 11.1 # nRBC 0.0 0.0 0.0 # nRBC 1.9 1.8 1.7 # nRBC0.3 0.3 0.3 % RETIC 5.15 4.36 4.06 % RETIC 1.89 2.10 1.84 % RETIC 0.020.03 0.00 # RETIC 0.13 0.11 0.10 # RETIC 0.08 0.09 0.08 # RETIC 0.000.00 0.00 % NEUT 59.8 59.0 61.3 % NEUT 60.7 59.5 61.9 % NEUT 59.6 59.159.4 # NEUT 11.12 11.38 11.58 # NEUT 5.58 5.46 5.65 # NEUT 1.62 1.691.58 % LYM 37.3 38.9 35.8 % LYM 36.2 37.1 35.0 % LYM 35.2 35.9 36.6 #LYM 6.94 7.51 6.76 # LYM 3.33 3.41 3.20 # LYM 0.95 1.03 0.95 % MONO 2.21.6 2.4 % MONO 2.3 3.0 2.5 % MONO 3.4 3.9 3.6 # MONO 0.41 0.30 0.46 #MONO 0.22 0.28 0.23 # MONO 0.09 0.11 0.09 % EOS 0.6 0.5 0.4 % EOS 0.50.2 0.4 % EOS 1.4 1.1 1.4 # EOS 0.11 0.09 0.08 # EOS 0.05 0.02 0.03 #EOS 0.04 0.03 0.04 % BASO 0.1 0.2 0.1 % BASO 0.2 0.2 0.2 % BASO 0.1 0.10.0 # BASO 0.02 0.03 0.02 # BASO 0.01 0.02 0.01 # BASO 0.00 0.00 0.00

For the above tables the following abbreviations are used: WBC: whiteblood cell; RBC: red blood cell; HGB: hemoglobin; HCT: hematocrit; MCV:mean corpuscular volume; MCH: mean corpuscular mass; MCHC: meancorpuscular hemoglobin concentration; PLT: platelet; RDW: red blood celldistribution width; SD RDW: standard deviation for red blood celldistribution width; MPV: mean platelet volume; nRBC: nucleated red bloodcell; RETIC: reticulocyte; NEUT: neutrophil; LYM: lymphocyte; MONO:monocyte; EOS: eosinophil; BASO: basophil.

General Remarks Applicable to All Teachings

As used herein, unless otherwise stated, the teachings envision that anymember of a genus (list) may be excluded from the genus; and/or anymember of a Markush grouping may be excluded from the grouping.

Unless otherwise stated, any numerical values recited herein include allvalues from the lower value to the upper value in increments of one unitprovided that there is a separation of at least 2 units between anylower value and any higher value. As an example, if it is stated thatthe amount of a component, a property, or a value of a process variablesuch as, for example, temperature, pressure, time and the like is, forexample, from 1 to 90, preferably from 20 to 80, more preferably from 30to 70, it is intended that intermediate range values such as (forexample, 15 to 85, 22 to 68, 43 to 51, 30 to 32 etc.) are within theteachings of this specification. Likewise, individual intermediatevalues are also within the present teachings. For values which are lessthan one, one unit is considered to be 0.0001, 0.001, 0.01 or 0.1 asappropriate. These are only examples of what is specifically intendedand all possible combinations of numerical values between the lowestvalue and the highest value enumerated are to be considered to beexpressly stated in this application in a similar manner. As can beseen, the comparative teaching of amounts expressed as weight/volumepercent for two or more ingredients also encompasses relative weightproportions of the two or more ingredients to each other, even if notexpressly stated. For example, if a teaching recites 2% A, and 5% B,then the teaching also encompasses a weight ratio of A:B of 2:5. Unlessotherwise stated, all ranges include both endpoints and all numbersbetween the endpoints. The use of “about” or “approximately” inconnection with a range applies to both ends of the range. Thus, “about20 to 30” is intended to cover “about 20 to about 30”, inclusive of atleast the specified endpoints.

The disclosures of all articles and references, including patentapplications and publications, are incorporated by reference for allpurposes.

The term “consisting essentially of to describe a combination shallinclude the elements, ingredients, components or steps identified, andsuch other elements ingredients, components or steps that do notmaterially affect the basic and novel characteristics of thecombination. The use of the terms “comprising” or “including” todescribe combinations of elements, ingredients, components or stepsherein also contemplates embodiments that consist essentially of(namely, the presence of any additional elements, ingredients,components or steps, does not materially affect the properties and/orbenefits derived from the teachings; or even consist of the elements,ingredients, components or steps.

Plural elements, ingredients, components or steps can be provided by asingle integrated element, ingredient, component or step. Alternatively,a single integrated element, ingredient, component or step might bedivided into separate plural elements, ingredients, components or steps.The disclosure of “a” or “one” to describe an element, ingredient,component or step is not intended to foreclose additional elements,ingredients, components or steps. All references herein to elements ormetals belonging to a certain Group refer to the Periodic Table of theElements published and copyrighted by CRC Press, Inc., 1989. Anyreference to the Group or Groups shall be to the Group or Groups asreflected in this Periodic Table of the Elements using the IUPAC systemfor numbering groups.

It is understood that the above description is intended to beillustrative and not restrictive. Many embodiments as well as manyapplications besides the examples provided will be apparent to those ofskill in the art upon reading the above description. The scope of theteachings should, therefore, be determined not with reference to theabove description, but should instead be determined with reference tothe appended claims, along with the full scope of equivalents to whichsuch claims are entitled. The disclosures of all articles andreferences, including patent applications and publications, areincorporated by reference for all purposes. The omission in thefollowing claims of any aspect of subject matter that is disclosedherein is not a disclaimer of such subject matter, nor should it beregarded that the inventors did not consider such subject matter to bepart of the disclosed inventive subject matter.

The explanations and illustrations presented herein are intended toacquaint others skilled in the art with the teachings, its principles,and its practical application. Those skilled in the art may adapt andapply the teachings in its numerous forms, as may be best suited to therequirements of a particular use. Accordingly, the specific embodimentsof the present teachings as set forth are not intended as beingexhaustive or limiting of the teachings. The scope of the teachingsshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to theappended claims, along with the full scope of equivalents to which suchclaims are entitled. The disclosures of all articles and references,including patent applications and publications, are incorporated byreference for all purposes. Other combinations are also possible as willbe gleaned from the following claims, which are also hereby incorporatedby reference into this written description.

The invention claimed is:
 1. A suspension composition comprising: one ormore simulated blood components; a buffered aqueous solution; and atleast one stabilizing agent, wherein the at least one stabilizing agentincludes one or any combination of chitin, a salt of chitin, aderivative of chitin, a chitosan, a salt of a chitosan, or a derivativeof a chitosan, and wherein the one or more simulated blood componentsare derived from blood cells processed to preserve characteristicsdetectable by a hematology analyzer.
 2. The suspension composition ofclaim 1, wherein the at least one stabilizing agent is present in anamount sufficient for preserving stability of detectable morphologicalcharacteristics of the one or more simulated blood components for aperiod of at least 30 days, after being stored during such period atabout 2 to about 10° C., upon suspending the blood components; whereinthe one or more simulated blood components include nucleated bloodcells, and wherein the detectable morphological characteristics that arepreserved include size, a nucleus morphology and morphology of othernative nuclear cytoplasm granules.
 3. The suspension composition ofclaim 1, wherein the aqueous buffered solution includes at least onebuffering agent, and at least one antimicrobial.
 4. The suspensioncomposition of claim 1, wherein the aqueous buffered solution includesat least one dispersion agent for reducing aggregation of the one ormore simulated blood components as compared with the aqueous bufferedsolution without the dispersion agent.
 5. The suspension composition ofclaim 1, wherein the suspension composition has a pH ranging from about6 to about
 8. 6. The suspension composition of claim 1, wherein the atleast one stabilizing agent is selected from chitosan acetate, chitosanlactate, chitosan oligosaccharide, carboxyl methyl chitosan, or anycombination thereof.
 7. The suspension composition of claim 1, whereinthe at least one stabilizing agent is present in an amount up to aboutten percent (10%) (weight/volume) of the suspension composition.
 8. Thesuspension composition of claim 1, wherein the one or more simulatedblood components includes simulated nucleated leukocytes, nucleated redblood cells or both.
 9. The suspension composition of claim 1, whereinthe suspension composition and any resulting control compositionemploying the suspension composition is substantially free of any addedlipoprotein.
 10. A method of using the suspension composition of claim1, comprising dispensing the suspension composition onto a substrate,and at least partially evaporating water from the aqueous bufferedsuspension composition.
 11. The suspension composition of claim 1,wherein the at least one stabilizing agent is present in an amount up toabout seven percent (7%) (weight/volume) of the suspension composition.12. The suspension composition of claim 1, wherein the at least onestabilizing agent is present in an amount of about five percent (5%)(weight/volume) of the suspension composition.
 13. The suspensioncomposition of claim 1, wherein the one or more simulated bloodcomponents comprises one or any combination of components for simulatinga platelet, a reticulated platelet, a red blood cell, reticulocyte, animmature reticulocyte, a nucleated red blood cell, or a simulatedleukocyte population or sub-population.
 14. The suspension compositionof claim 1, wherein the amount of the one or more simulated bloodcomponents is a known amount for simulating a normal amount in wholeblood.
 15. The method of claim 10, further comprising creating andanalyzing an image of the suspension composition in a digital imaginghematology instrument.
 16. The suspension composition of claim 1,wherein the at least one stabilizing agent is present in an amountsufficient for preserving stability of detectable morphologicalcharacteristics of the one or more simulated blood components for aperiod of at least 3 days, after being stored during such period atabout 1 to about 10° C., upon suspending the one or more simulated bloodcomponents.
 17. The suspension composition of claim 1, wherein the oneor more simulated blood components comprises neutrophils, lymphocytes,and monocytes.
 18. The suspension composition of claim 1, wherein theone or more simulated blood components comprises neutrophils,eosinophils, basophils, lymphocytes, and monocytes.